Mammalian tissue factor is a plasma membrane glycoprotein that binds to blood coagulation factor VII or factor VIIIa. The binding activity of tissue factor is the initiating event in the cascade of enzymatic activities which lead to blood coagulation and clot formation in mammals. Tissue factor is found in many cell types that are not exposed directly to flowing blood and is particularly abundant in the brain, lung, and placenta. Injuries that disrupt the endothelium bring factor VII into contact with tissue factor to initiate the cascade that leads to blood clotting and clot formation.
Protein tissue factor is a valuable biological commodity in commerce today. The main use of tissue factor is as a constituent of the standard blood clotting test procedure performed on patients prior to surgery. The coagulation test most commonly used is initiated by the interaction of tissue factor in vitro to other blood coagulation factors such as factor VII. Such in vitro tests, in several forms, are marketed by several companies throughout the world. One category of such tests is referred to as the prothrombin time (PT) test in which a series of enzymatic tests in vitro under controlled conditions is used to diagnose disfunctions in the blood coagulation system of patients. Up until recently, in all commercially available PT tests the PT reagents included a relatively crude tissue factor protein extracted from natural sources, typically rabbits. Rabbit brain and rabbit lung mixtures were subjected to a series of isolation and purification steps, resulting in a dried extract of rabbit tissue factor used commercially in PT kits. While the use of rabbit tissue factor from natural sources was practical and reasonably well defined, there was occasional variation in the activity and yield of rabbit tissue factor from those preparations, both due to seasonable variability in the abundance of tissue factor in rabbits as well as lot-to-lot variability due to normal variation of biological systems. Such tissue factor extracts could also come on occasion, containing other coagulation factors in small amounts and a variety of other poorly characterized constituents.
Accordingly, significant effort was extended toward cloning and isolating tissue factor for use in the recombinant production of tissue factor as a protein. This led to the cloning of human tissue factor from a variety of cDNA segments as described in U.S. Pat. No. 5,110,730. The purification and preparation of prothrombin time reagents from such recombinant human tissue factor is described in published PCT application WO 93/07492. Products have been introduced now into commerce based on the production and purification of recombinant human tissue factor. The methods used to produce recombinant human tissue factor have not been fully elucidated in the literature.
The cDNA of rabbit brain tissue factor has also been isolated and sequenced. Andrews et al., Gene, 96:265-269 (1991); Pawashe et al., Thrombosis and Haemostasis 66:3:315-320 (1991).
Using current techniques of modern molecular biology or genetic engineering, it has now become commonplace to produce therapeutic proteins in prokaryotic hosts reproduced in fermenters or other vessels. However, experience has revealed that certain proteins are more amenable to production in this fashion than others. For example, proteins which have a relatively large number of cysteines, which form disulfide bridges with other cysteines in a protein molecule, are often found to be inefficiently produced in a biologically active form in a prokaryotic host. The reason is that the protein assembly and processing machinery of prokaryotes differ significantly from that present in eukaryotes. If improper disulfide bridges are formed between the cysteine molecules of a protein, it will not be biologically active. Accordingly, proteins with many disulfide bonds are often difficult to be produced in commercially significant quantities in prokaryotic systems and thus are sometimes produced in eukaryotic host systems. This is less desirable commercially since the culture and fermentation facilities for eukaryotic cell culture systems are currently much more expensive than those required for prokaryotic protein production hosts.
Another difficulty in producing therapeutic proteins in prokaryotic hosts arises with proteins which are particularly hydrophobic or have strong hydrophobic regions. This difficulty arises because of solubility problems in the protein produced with the host. Poorly soluble proteins can be difficult to recover in economic quantities when expressed in prokaryotic host expression systems.
Native rabbit tissue factor is, like the human tissue factor, a membrane-bound protein containing three domains. One domain is an extracellular domain of 217 to 222 amino acids. There is also a hydrophobic putative transmembrane domain of 23 amino acids and a cytoplasmic domain of 20 to 21 amino acids. Thus the protein produced by the cloned rabbit tissue factor gene has a large and quite hydrophobic domain. In addition, there are four conserved cysteine residues in all known tissue factor sequences and the proper formation of the cysteine disulfide bonds is required to have proper biological activity for the tissue factor protein.